Antibody-containing solution pharmaceuticals

ABSTRACT

Antibody-containing solution formulations including a sugar as a stabilizer. Said solution formulations can further include a surfactant as a stabilizer.

TECHNICAL FIELD

The present invention relates to stable antibody-containing solutionformulations.

BACKGROUND ART

With the development of genetic engineering technology, it has becomepossible to use antibodies such as immunoglobulins, monoclonalantibodies and humanized antibodies as pharmaceutical products. Tosupply them in stable amounts, it is necessary to establish preparationconditions and storage conditions under which the structure and activityof the antibodies can be retained.

When proteins are stored in high concentration solutions, they normallysuffer deterioration such as the formation of insoluble aggregates,which must be prevented. Especially, antibody formulations have thedisadvantage that they tend to form multimers leading to insolubleaggregates during storage in solutions.

For example, we found that anti-IL-6 receptor antibodies have atherapeutic effect on immature myeloma cells (JPA HEI 8-99902) andsucceeded in mass-producing a reshaped humanized antibody, hPM-1antibody, as an anti-IL-6 receptor antibody, and we have tried toformulate this purified anti-IL-6 receptor antibody into pharmaceuticalproducts. The humanized anti-IL-6 receptor antibody is an unstableprotein liable to physical or chemical changes such as association oraggregation under the stresses of filtration, concentration, heat andlight for removing viruses and other microbials during purificationprocesses.

When antibodies are to be obtained by genetic engineering techniques,antibody-producing cells are cultured in bulk and purified to give anantibody-containing solution, which is then stored frozen and thawedbefore formulation. However, the antibody content remaining in such asolution decreased as antibody dimers or insoluble particles were formedduring repeated freeze/thaw cycles or antibodies were degraded to formdegradation products during long-term storage.

Many efforts have been made to provide a method for storing proteins insolutions, and a stabilization effect was found by adding polymersincluding proteins such as human serum albumin or purified gelatin oroligomers such as polyols, amino acids and surfactants as stabilizersfor preventing chemical or physical changes. However, the addition ofbiopolymers such as proteins as stabilizers was inconvenient, e.g. itrequired a very complicated step for eliminating contaminants such asviruses and prions. As to the addition of oligomers, it shouldpreferably be minimized.

Freeze-dried antibody formulations stabilized with sugars or aminosugars, amino acids and surfactants have also been reported (JPAHEI2001-503781).

However, stable antibody-containing solution formulations have beensought because of great demands for easy-to-use solution formulationsthat may not be dissolved/reconstituted before use.

DISCLOSURE OF THE INVENTION

An object of the present invention is to provide antibody-containingsolution formulations in which the antibody content remains high, andwhich are stable even after long-term storage by inhibiting theformation of insoluble particles and multimers during the preparation orstorage of the antibody-containing solution formulations and furtherinhibiting the formation of degradation products.

As a result of careful studies to attain the above object, weaccomplished the present invention on the basis of the finding that theformation of dimers during freeze/thaw cycles or the formation ofmultimers and degradation products during long-term storage can beinhibited by adding a sugar, and that the formation of insolubleparticles during freeze/thaw cycles can be remarkably inhibited byadding a surfactant.

Accordingly, the present invention provides:

(1) an antibody-containing solution formulation including a sugar as astabilizer;

(2) the solution formulation as defined in (1) further including asurfactant as a stabilizer;

(3) the solution formulation as defined in (1) or (2) wherein the sugaris a sugar alcohol or a nonreducing oligosaccharide;

(4) the solution formulation as defined in (1) or (2) wherein the sugaris a nonreducing oligosaccharide;

(5) the solution formulation as defined in (1) or (2) wherein the sugaris mannitol, sucrose, trehalose or raffinose;

(6) the solution formulation as defined in (1) or (2) wherein the sugaris sucrose, trehalose or raffinose;

(7) the solution formulation as defined in (1) or (2) wherein the sugaris sucrose or trehalose;

(8) the solution formulation as defined in (1) or (2) wherein the sugaris sucrose;

(9) the solution formulation as defined in any one of (2) to (8) whereinthe surfactant is Polysorbate 80 or 20;

(10) the solution formulation as defined in any one of (1) to (9)wherein the antibody is a recombinant antibody;

(11) the solution formulation as defined in (10) wherein the antibody isa chimeric antibody, humanized antibody or human antibody;

(12) the solution formulation as defined in any one of (1) to (11)wherein the antibody is an IgG class antibody;

(13) the solution formulation as defined in (12) wherein the IgG classantibody is an IgGl class antibody;

(14) the solution formulation as defined in any one of (1) to (13)wherein the antibody is an anti-interleukin-6 receptor antibody oranti-HM1.24 antibody;

(15) a method for inhibiting the formation of antibody multimermolecules in an antibody-containing solution formulation, comprisingadding a sugar to the solution;

(16) a method for inhibiting the formation of antibody multimermolecules during freeze/thaw cycles of an antibody-containing solution,comprising adding a nonreducing oligosaccharide to the solution;

(17) a method for inhibiting the formation of antibody multimermolecules during freeze/thaw cycles of an antibody-containing solution,comprising adding a nonreducing disaccharide or nonreducingtrisaccharide into the solution;

(18) a method for inhibiting the formation of insoluble particles duringfreeze/thaw cycles of an antibody-containing solution, comprising addinga surfactant; and

(19) a method for stabilizing an antibody during freeze/thaw cycles of asolution containing the antibody, comprising adding a nonreducing sugarand a surfactant.

THE MOST PREFERRED EMBODIMENTS OF THE INVENTION

As used herein, “antibody-containing solution formulation” means asolution formulation containing an antibody as an active ingredient andprepared for administration to animals such as humans, preferablywithout including any freeze-drying steps in the preparation process.

As used herein, “antibody-containing solution” may be a solutioncontaining any antibody, whether biologically derived or recombinant,preferably a culture medium in which mammalian cells such as CHO cellscontaining an antibody have been cultured, or a solution obtained bysubjecting such a medium to a given treatment such as partialpurification (bulk solution), or the solution formulation prepared foradministration to animals such as humans as defined above.

As used herein, the term “insoluble particles” means insolubleparticulate matters of 10 μm or more as defined in the section ofInsoluble Particulate Matter Test for Injections in the part of GeneralTests, Processes and Apparatus in the Japanese Pharmacopoeia. Insolubleparticles can be measured by using microscopes, insolubleparticle-collecting filters and analytical membrane filters, orconveniently using automatic light obscuration particle counters.

As used herein, “insoluble matters” mean readily detectable insolublematters from which injections must be free and clear when inspected incontainers with the unaided eye with a light intensity of approximately1000 luxes under an incandescent lamp as defined in the section ofForeign Insoluble Matter Test for Injections in the part of GeneralTests, Processes and Apparatus in the Japanese Pharmacopoeia.

As used herein, “multimers” and “degradation products” mean multimersand degradation products respectively of antibody molecules constitutingactive ingredients of formulations, and their contents can be determinedby the peak area percentage method based on gel permeationchromatography described later.

Antibodies used in solution formulations of the present invention arenot specifically limited so far as they bind to a desired antigen, andmouse antibodies, rat antibodies, rabbit antibodies, sheep antibodies,chimeric antibodies, humanized antibodies, human antibodies and the likecan be used as appropriate. The antibodies may be polyclonal ormonoclonal, but preferably monoclonal because homogeneous antibodies canbe stably produced. Polyclonal and monoclonal antibodies can be preparedby processes well known to those skilled in the art.

Hybridomas producing monoclonal antibodies can be basically constructedby known techniques as follows. A desired antigen or a cell expressing adesired antigen is used as an immunizing antigen to immunize host cellsaccording to a standard immunization technique, and the resultingimmunized cells are fused to known parent cells by a standard cellfusion technique, and then the fused cells are screened for monoclonalantibody-producing cells (hybridomas) by a standard screening method.Construction of hybridomas can be performed according to the method ofe.g. Milstein et al. (Kohler. G. and Milstein, C., Methods Enzymol.(1981) 73: 3-46). If the antigen has low immunogenicity, it can be boundto an immunogenic macromolecule such as albumin and used forimmunization.

Recombinant antibodies can be used, which are produced by transforming ahost with a suitable vector containing an antibody gene cloned from ahybridoma using genetic engineering techniques (see e.g. Carl, A. K.Borrebaeck, James, W. Larrick, THERAPEUTIC MONOCLONAL ANTIBODIES,Published in the United Kingdom by MACMILLAN PUBLISHERS LTD, 1990).Specifically, the cDNA sequences for the variable regions (V regions) ofan antibody are synthesized from mRNA of a hybridoma using a reversetranscriptase. Once DNA sequences encoding the V regions of the antibodyof interest have been obtained, they may be linked to the DNA sequencesencoding the constant regions (C regions) of the antibody of interestand integrated into an expression vector. Alternatively, the DNAsequences encoding the V regions of the antibody may be integrated intoan expression vector containing the DNA sequences for the C regions ofthe antibody. They are integrated into the expression vector in such amanner that they can be expressed under the control of regulatoryregions such as enhancers and promoters. Then, a host cell can betransformed with this expression vector to express the antibody.

In the present invention, recombinant antibodies, i.e. antibodiesartificially modified to reduce antigenicity in humans or to attainother purposes, such as chimeric antibodies and humanized antibodies canbe used. These modified antibodies can be prepared by known processes.Chimeric antibodies consist of the heavy and light chain variableregions of an antibody from a non-human mammal such as a mouse and theheavy and light chain constant regions of a human antibody and can beobtained by linking the DNA sequences encoding the variable regions ofthe mouse antibody to the DNA sequences for the constant regions of thehuman antibody and transforming a host with an expression vectorcontaining the linked sequences to allow it to produce a chimericantibody.

Humanized antibodies are also called reshaped human antibodies andobtained by grafting the complementarity-determining regions (CDRs) ofan antibody from a non-human mammal such as a mouse into thecomplementarity-determining regions of a human antibody and typical generecombination techniques for preparing them are also known.Specifically, DNA sequences designed to link the CDRs of a mouseantibody to the framework regions (FRs) of a human antibody aresynthesized by PCR from several oligonucleotides prepared to haveterminal overlapping regions. The resulting DNA sequences are linked tothe DNA sequences encoding the constant regions of the human antibodyand then integrated into an expression vector, which is transformed intoa host to allow it to produce a reshaped antibody (see European PatentPublication No. EP 239400, International Publication No. WO 96/02576).The FRs of the human antibody linked by the CDRs are selected in such amanner that the complementarity-determining regions form an appropriateantigen-binding site. If necessary, reshaped humanized antibodies mayhave some amino acid changes in the framework regions of the variableregions so that the complementarity-determining regions form anappropriate antigen-binding site (Sato, K. et al., Cancer Res. (1993)53, 851-856).

Methods for obtaining human antibodies are also known. For example, adesired human antibody having a binding activity for a desired antigencan be obtained by in vitro immunizing human lymphocytes with thedesired antigen or a cell expressing the desired antigen and fusing theimmunized lymphocytes to human myeloma cells such as U266 (see JPB No.HEI1-59878). A desired human antibody can also be obtained by immunizinga transgenic animal having all human antibody gene repertoires with anantigen (see International Publications Nos. WO 93/12227, WO 92/03918,WO 94/02602, WO 94/25585, WO 96/34096, WO 96/33735). Methods forobtaining a human antibody by panning using a human antibody library arealso known. For example, phages binding to an antigen can be selected byexpressing the variable regions of a human antibody as single chainantibody fragments (scFv) on phage surfaces by a phage display method.The DNA sequences encoding the variable regions of the human antibodybinding to the antigen can be determined by analyzing the genes of theselected phages. A whole human antibody can be obtained by preparing asuitable expression vector on the basis of the determined DNA sequencesof the scFv fragments binding to the antigen. These methods are alreadywell known from WO 92/01047, WO 92/20791, WO 93/06213, WO 93/11236, WO93/19172, WO 95/01438, WO 95/15388.

When an antibody is to be prepared by transforming a preliminarilyisolated antibody gene into a suitable host, a suitable host can be usedin combination with an expression vector. Suitable eukaryotic cells usedas hosts include animal cells, plant cells and fungal cells. Knownanimal cells include (1) mammal cells such as CHO, COS, myeloma, BHK(baby hamster kidney), HeLa and Vero cells; (2) amphibian cells such asXenopus oocytes; or (3) insect sells such as sf9, sf21 and Tn5. Knownplant cells include cells of Nicotiana such as Nicotiana tabacum, whichcan be used as callus cultures. Known fungal cells include yeasts suchas Saccharomyces spp., e.g. Saccharomyces serevisiae and filamentousfungi such as Aspergillus spp., e.g. Aspergillus niger. Prokaryoticcells can be used as producing systems using bacterial cells. Knownbacterial cells include E. coli and Bacillus subtilis. Antibodies can beobtained by transforming these cells with an antibody gene of interestand culturing the transformed cells in vitro.

Antibodies contained in stabilized formulations of the present inventioninclude, but not limited to, anti-IL-6 receptor antibodies, anti-HM1.24antigen monoclonal antibodies, anti-parathyroid hormone related peptideantibodies (anti-PTHrP antibodies), etc.

Preferred reshaped humanized antibodies for use in the present inventioninclude humanized anti-IL-6 receptor antibodies (hPM-1) (seeInternational Publication No. W092-19759), humanized anti-HM1.24 antigenmonoclonal antibodies (see International Publication No. W098-14580) andhumanized anti-parathyroid hormone related peptide antibodies(anti-PTHrP antibodies) (see International Publication No. W098-13388).

Antibodies contained in solution formulations of the present inventionmay belong to any immunoglobulin class, preferably IgG such as IgG1,IgG2, IgG3 and IgG4, more preferably IgG1.

Antibody-containing solution formulations in the present inventionpreferably show no increase in multimers and contain 50 or lessinsoluble particles per mL after freeze/thaw cycling.

In antibody-containing solutions or solution formulations of the presentinvention, the formation of dimers during freeze/thaw cycles can beinhibited by adding sugars. The sugars that can be used includenonreducing oligosaccharides, e.g. nonreducing disaccharides such assucrose and trehalose or nonreducing trisaccharides such as raffinose,and especially preferred are nonreducing oligosaccharides. Preferrednonreducing oligosaccharides are nonreducing disaccharides, morepreferably sucrose and trehalose.

In antibody-containing solutions or solution formulations of the presentinvention, the formation of multimers and degradation products duringlong-term storage can be inhibited by adding sugars. The sugars that canbe used include sugar alcohols such as mannitol and sorbitol; andnonreducing oligosaccharides, e.g. nonreducing disaccharides such assucrose and trehalose or nonreducing trisaccharides such as raffinose,among which nonreducing oligosaccharides are especially preferred.Preferred nonreducing oligosaccharides are nonreducing disaccharides,more preferably sucrose and trehalose.

The sugars should be added at 0.1-500 mg/mL, preferably 10-300 mg/mL,more preferably 25-100 mg/mL.

In the present invention, the formation of insoluble particles duringfreeze/thaw cycles of antibody-containing solution formulations can bevery remarkably inhibited by adding surfactants. Typical examples ofsurfactants include:

-   -   nonionic surfactants, e.g., sorbitan fatty acid esters such as        sorbitan monocaprylate, sorbitan monolaurate, sorbitan        monopalmitate; glycerin fatty acid esters such as glycerin        monocaprylate, glycerin monomyristate, glycerin monostearate;        polyglycerin fatty acid esters such as decaglyceryl        monostearate, decaglyceryl distearate, decaglyceryl        monolinoleate; polyoxyethylene sorbitan fatty acid esters such        as polyoxyethylene sorbitan monolaurate, polyoxyethylene        sorbitan monooleate, polyoxyethylene sorbitan monostearate,        polyoxyethylene sorbitan monopalmitate, polyoxyethylene sorbitan        trioleate, polyoxyethylene sorbitan tristearate; polyoxyethylene        sorbitol fatty acid esters such as polyoxyethylene sorbitol        tetrastearate, polyoxyethylene sorbitol tetraoleate;        polyoxyethylene glycerin fatty acid esters such as        polyoxyethylene glyceryl monostearate; polyethylene glycol fatty        acid esters such as polyethylene glycol distearate;        polyoxyethylene alkyl ethers such as polyoxyethylene lauryl        ether; polyoxyethylene polyoxypropylene alkyl ethers such as        polyoxyethylene polyoxypropylene glycol ether, polyoxyethylene        polyoxypropylene propyl ether, polyoxyethylene polyoxypropylene        cetyl ether; polyoxyethylene alkyl phenyl ethers such as        pblyoxyethylene nonyl phenyl ether; polyoxyethylene hardened        castor oils such as polyoxyethylene castor oil, polyoxyethylene        hardened castor oil (polyoxyethylene hydrogenated castor oil);        polyoxyethylene beeswax derivatives such as polyoxyethylene        sorbitol beeswax; polyoxyethylene lanolin derivatives such as        polyoxyethylene lanolin; polyoxyethylene fatty acid amides such        as polyoxyethylene stearic acid amide having an HLB of 6-18;    -   anionic surfactants, e.g., alkyl sulfates having a C10-18 alkyl        group such as sodium cetyl sulfate, sodium lauryl sulfate,        sodium oleyl sulfate; polyoxyethylene alkyl ether sulfates        having an average EO mole number of 2-4 and a C10-18 alkyl group        such as sodium polyoxyethylene lauryl sulfate; alkyl        sulfosuccinic acid ester salts having a C8-18 alkyl group such        as sodium laurylsulfosuccinate; and    -   natural surfactants, e.g., lecithin; glycerophospholipids;        sphingophospholipids such as sphingomyelin; sucrose fatty acid        esters of C12-18 fatty acids. Formulations of the present        invention can contain one or more of these surfactants in        combination. Preferred surfactants for use in solution        formulations of the present invention are polyoxyethylene        sorbitan fatty acid esters such as Polysorbate 20, 40, 60 or 80,        especially Polysorbates 20 and 80. Polyoxyethylene        polyoxypropylene glycols such as poloxamers (e.g. Pluronic®        F-68) are also preferred.

The amount of surfactants to be added varies with the type of theparticular surfactant used, but it is typically 0.001-100 mg/mL,preferably 0.003-50 mg/mL, more preferably 0.005-2 mg/mL in the case ofPolysorbate 20 or Polysorbate 80.

Preferably, antibody-containing solution formulations of the presentinvention are substantially free from proteins such as human serumalbumin or purified gelatin as stabilizers.

Antibody formulations of the present invention preferably have a pH of4-8, more preferably 5-7, still more preferably 6-6.5. However, the pHdepends on the antibody contained and is not limited to these values.

Formulations of the present invention may further contain isotonizingagents, e.g., polyethylene glycol; and sugars such as dextran, mannitol,sorbitol, inositol, glucose, fructose, lactose, xylose, mannose,maltose, sucrose, trehalose and raffinose.

Antibody-containing solution formulations of the present invention mayfurther contain diluents, solubilizing agents, excipients, pH-modifiers,soothing agents, buffers, sulfur-containing reducing agents,antioxidants or the like, if desired. For example, sulfur-containingreducing agents include N-acetylcysteine, N-acetylhomocysteine, thiocticacid, thiodiglycol, thioethanolamine, thioglycerol, thiosorbitol,thioglycolic acid and salts thereof, sodium thiosulfate, glutathione,and sulfhydryl-containing compounds such as thioalkanoic acid having 1to 7 carbon atoms. Antioxidants include erythorbic acid,dibutylhydroxytoluene, butylhydroxyanisole, α-tocopherol, tocopherolacetate, L-ascorbic acid and salts thereof, L-ascorbyl palmitate,L-ascorbyl stearate, sodium bisulfite, sodium sulfite, triamyl gallate,propyl gallate or chelating agents such as disodium ethylenediaminetetraacetate (EDTA), sodium pyrophosphate and sodium metaphosphate.Other common additives may also be contained, e.g., inorganic salts suchas sodium chloride, potassium chloride, calcium chloride, sodiumphosphate, potassium phosphate and sodium bicarbonate; and organic saltssuch as sodium citrate, potassium citrate and sodium acetate.

Formulations of the present invention can be prepared by dissolvingthese components in an aqueous buffer known in the field of solutionformulations such as a phosphate buffer (preferably sodium monohydrogenphosphate—sodium dihydrogen phosphate system) and/or a citrate buffer(preferably sodium citrate buffer) and/or an acetate buffer to prepare asolution formulation. The concentration of the buffer is typically 1-500mM, preferably 5-100 mM, more preferably 10-20 mM.

Antibody-containing solution formulations of the present invention arenormally administered via parenteral routes such as injection (e.g.subcutaneous, intravenous, intramuscular or intraperitoneal injection)or percutaneous, mucosal, nasal or pulmonary administration, but mayalso be orally administered.

Antibody-containing solution formulations of the present invention canbe normally supplied in sealed and sterilized plastic or glasscontainers having a defined volume such as vials, ampules or syringes ora large volume such as bottles. In terms of convenience, prefilledsyringes are preferred.

The amount of antibodies contained in formulations of the presentinvention is typically 0.1-200 mg/ml, preferably 1-120 mg/ml, morepreferably 2-22.5 mg/mL, depending on the type of the disease to betreated, the severity of the disease, the age of the patient and otherfactors.

In solution formulations of the present invention, the formation ofinsoluble particles especially during freeze/thaw cycles could beremarkably inhibited and the formation of insoluble matters duringlong-term stability tests could also be remarkably inhibited by addingsurfactants, as shown in the examples below. It was also found that theformation of multimers such as dimers as well as the formation ofdegradation products could be remarkably inhibited and remainingantibody monomer contents could be increased by adding sugars.

The following examples further illustrate the present invention without,however, limiting the scope of the invention thereto. Various changesand modifications can be made by those skilled in the art on the basisof the description of the invention, and such changes and modificationsare also included in the present invention.

EXAMPLES

Antibody Samples

An hPM-1 antibody was used as a humanized anti-IL-6 receptor antibody.The hPM-1 antibody was a humanized hPM-1 antibody prepared by the methoddescribed in Reference example 2 of JPA HEI 8-99902 using the humanelongation factor Iα promoter described in Example 10 of InternationalPatent Publication No. WO92/19759.

An antibody prepared by the method described in Reference example 2 ofInternational Patent Publication No. WO98-35698 (hereinafter referred toas anti-HM1.24 antibody) was used as a humanized anti-HM1.24 antigenmonoclonal antibody.

The hPM-1 antibody and anti-HM1.24 antibody used in the followingexamples are both IgG1 class antibodies.

Test Methods

(A) Tests on hPM-1 antibody

(1) Gel permeation chromatography (GPC)

Each sample is diluted with the mobile phase to contain hPM-1 in anamount equivalent to about 1 mg in 1 mL and tested under the followingHPLC conditions in 30-60 μL.

Column: TSK gel G3000SW_(XL) (TOSOH)

Guard column: TSK guard column SW_(XL) (TOSOH)

Column temperature: constant around 25° C.

Mobile phase: 50 mM phosphate buffer (pH 7.0)-300 mM sodium chloride

Flow rate: about 1.0 mL/min

Measured at wavelength: 280 nm.

The peak area was determined by automatic integration to calculate thehPM-1 content from the peak area of a standard hPM-1 product and theremaining hPM-1 percentage from the initial evaluation results using thefollowing equations.${{hPM}\text{-}1\quad{content}\quad( {{mg}\text{/}{mL}} )} = \frac{\begin{matrix}{{Concentration}\quad{of}\quad{standard}} \\{{hPM}\text{-}1\quad{product} \times {Peak}} \\{{area}\quad{of}\quad{test}\quad{sample}}\end{matrix}}{{Peak}\quad{area}\quad{of}\quad{standard}\quad{hPM}\text{-}1\quad{product}}$${{Remaining}\quad{hPM}\text{-}1\quad{percentage}\quad(\%)} = {\frac{\begin{matrix}{{hPM}\text{-}1\quad{content}\quad{after}\quad{thermal}} \\{{acceleration}\quad{and}\quad{{freeze}/{thaw}}\quad{cycles}}\end{matrix}}{{Initial}\quad{hPM}\text{-}1\quad{content}} \times 100}$

The percentages of dimers, other multimers and degradation products werecalculated by the area percentage method using the following equation.$\begin{matrix}{{Dimers}\quad( {{or}\quad{other}\quad{multimers}} } \\{ \quad{{or}\quad{degredation}\quad{products}} )\quad(\%)}\end{matrix} = {\frac{\begin{matrix}{{Peak}\quad{area}\quad{of}\quad{dimers}} \\( {{or}\quad{other}\quad{multimers}\quad{or}\quad{degradation}\quad{products}} )\end{matrix}}{{Total}\quad{peak}\quad{area}} \times 100}$

(2) Evaluation of the number of insoluble particles by a lightobscuration automatic particle counter (HIAC)

Evaluation was made according to the method using an automatic lightobscuration particle counter as described in the section of InsolubleParticulate Matter Test for Injections in the part of General Tests,Processes and Apparatus in the Japanese Pharmacopoeia.

(3) Automated visual inspection

Automated visual inspection was performed according to the method asdescribed in the section of Foreign Insoluble Matter Test for Injectionsin the part of General Tests, Processes and Apparatus in the JapanesePharmacopoeia.

Visual inspection system: Type E422 (Eisai).

(B) Tests on anti-HM1.24 antibody

(1) Gel permeation chromatography (GPC); measured at N=3 to evaluate theremaining percentage (%) to the initial content and also evaluatemultimers and degradation products in percentages.

Column: TSK gel G3000SW_(XL) (TOSOH)

Guard column: TSK guard column SW_(XL) (TOSOH)

Column temperature: constant around 25° C.

Mobile phase: 50 mM phosphate buffer (pH 7.0)-300 mM sodium chloride

Flow rate: about 0.5 mL/min

Measured at wavelength: 280 nmMethod for Calculating the Concentration $\begin{matrix}{{Anti}\text{-}{HM1}{.24}\quad{antibody}} \\{{content}\quad( {{mg}\text{/}{mL}} )}\end{matrix} = \frac{\begin{matrix}{{Concentration}\quad{of}\quad{standard} \times} \\{{Peak}\quad{area}\quad{of}\quad{anti}\text{-}{HM1}{.24}\quad{antibody} \times} \\{{Amount}\quad{of}\quad{standard}\quad{applied}}\end{matrix}}{\begin{matrix}{{Total}\quad{peak}\quad{area}\quad{of}\quad{standard} \times} \\{{Amount}\quad{of}\quad{test}\quad{sample}\quad{applied}}\end{matrix}}$ $\begin{matrix}{{Remaining}\quad{anti}\text{-}{HM1}{.24}} \\{{antibody}\quad{percentage}\quad(\%)}\end{matrix} = {\frac{\begin{matrix}{{Anti}\text{-}{HM1}{.24}\quad{antibody}\quad{content}\quad{after}} \\{{thermal}\quad{acceleration}}\end{matrix}}{{Initial}\quad{anti}\text{-}{HM1}{.24}\quad{antibody}\quad{content}} \times 100}$

The percentages of multimers and degradation products were calculated bythe area percentage method.${{Multimers}\quad( {{or}\quad{degradation}\quad{products}} )\quad(\%)} = {\frac{\begin{matrix}{{Peak}\quad{area}\quad{of}\quad{multimers}} \\( {{or}\quad{degradation}\quad{products}} )\end{matrix}}{{Total}\quad{peak}\quad{area}} \times 100}$

Example 1 Effects of Adding A Surfactant (1)

The influence of a surfactant (Polysorbate 80) on heat stability andfreeze/thaw stability was tested. Samples containing Polysorbate 80 atvarious concentrations shown in Table 1 were prepared and tested asfollows.

(1) Stability to thermal acceleration (50° C.-2W) was evaluated from theremaining hPM-1 percentage and the formation of multimers anddegradation products as determined by gel permeation chromatography(GPC). The number of insoluble particles per mL was measured by anautomatic light obscuration particle counter (HIAC).

(2) Stability to freeze/thaw cycling (3 cycles of storage at −20° C. for3 days and then 5° C. for one day) was evaluated from the remaininghPM-1 percentage and the formation of multimers and degradation productsas determined by gel permeation chromatography (GPC). The number ofinsoluble particles per mL was measured by an automatic lightobscuration particle counter (HIAC).

The results obtained are shown in Table 1. TABLE 1 <Test samples andresults> Sample 1 Sample 2 Sample 3 Sample 4 hPM-1 (mg/mL) 20 20 20 20Polysorbate 80 (mg/mL) 0 0.25 0.5 0.75 Sodium Phosphate (mM) 15 15 15 15pH 6.5 6.5 6.5 6.5 Initial hPM-1 content (mg/mL) 20.1 20.3 20.3 20.4Dimers (%) 0.21 0.22 0.22 0.23 Other multimers (%) 0 0 0 0 Degradationproducts 0 0 0 0 (%) Number of particles 0 0 2 0 of 10 μm or more(particles/mL) Number of particles 0 0 0 0 of 25 μm or more(particles/mL) Thermal Remaining hPM-1 (%) 99.4 98.2 98.1 98.0acceleration Dimers (%) 1.38 1.39 1.39 1.41 (50° C.-2 W) Other multimers(%) 0 0 0 0 Degradation products 0.91 0.91 0.90 0.90 (%) Number ofparticles 0 0 0 0 of 10 μm or more (particles/mL) Number of particles 00 0 0 of 25 μm or more (particles/mL) Freeze/thaw Remaining hPM-1 (%)99.7 99.6 99.4 99.3 (−20° C.→5° C., Dimers (%) 0.60 0.56 0.52 0.49 3cycles) Other multimers (%) 0 0 0 0 Degradation products 0 0 0 0 (%)Number of particles 3287 7 1 4 of 10 μm or more (particles/mL) Number ofparticles 539 3 0 0 of 25 μm or more (particles/mL)

It was found that the formation of insoluble particles duringfreeze/thaw cycles is remarkably inhibited by the addition ofPolysorbate 80. No significant variation in stability with theconcentration of Polysorbate 80 was found.

Example 2 Effects of Adding A Surfactant (2)

The influence of a surfactant (Polysorbate 80) on stability tofreeze/thaw cycling and shaking was tested. Samples containingPolysorbate 80 at various concentrations shown in Table 2 were preparedand tested as follows.

Stability to freeze/thaw cycling (2 cycles of storage at −20° C. for 8hours and then 5° C. for 8 hours) was evaluated from the number ofinsoluble particles per mL as measured by an automatic light obscurationparticle counter (HIAC). The presence or absence of insoluble matterswas evaluated by automated visual inspection.

The results obtained are shown in Table 2. TABLE 2 <Test samples andresults> Sample 5 Sample 6 Sample 7 Sample 8 Sample 9 Sample 10 hPM-1(mg/mL) 20 20 20 20 20 20 Polysorbate 80 (mg/mL) 0 0.005 0.05 0.25 0.50.75 Sucrose (mg/mL) 50 50 50 50 50 50 Sodium Phosphate (mM) 15 15 15 1515 15 pH 6.5 6.5 6.5 6.5 6.5 6.5 Initial Number of 10 0 0 0 0 0particles of 10 μm or more (particles/mL) Number of 2 0 0 0 0 0particles of 25 μm or more (particles/mL) Insoluble Yes No No No No Nomatters Freeze/ Number of 7020 8 0 0 0 1 thaw particles of (−20° C.→5°C., 10 μm or more 2 cycles) (particles/mL) Number of 601 0 0 0 0 0particles of 25 μm or more (particles/mL) Insoluble Yes Yes No No No Nomatters

It was found that the formation of insoluble particles and insolublematters during freeze/thaw cycles is remarkably inhibited by theaddition of Polysorbate 80. The effect against the formation ofinsoluble matters was found to be dependent on the concentration ofPolysorbate 80.

Example 3 Effects of Adding Sugars

The influence of adding sugars on freeze/thaw stability was tested.Samples containing various sugars shown in Table 3 (sucrose, mannitol,trehalose) were prepared and evaluated for stability to freeze/thawcycling (22 cycles of storage at −20° C. for 2 hours and then 5° C. for2 hours) as determined by gel permeation chromatography (GPC) from theamount of dimers formed. TABLE 3 <Test samples and results> SampleSample Sample Sample Sample 11 12 13 14 15 hPM-1 (mg/mL) 20 20 20 20 20Sucrose (mg/mL) 0 50 0 0 0 Mannitol (mg/mL) 0 0 50 94 0 Trehalose (mg ·mL) 0 0 0 0 50 Polysorbate 80 (mg/mL) 0.5 0.5 0.5 0.5 0.5 SodiumPhosphate (mM) 15 15 15 15 15 pH 6.5 6.5 6.5 6.5 6.5 Initial Dimers 0.420.43 0.41 0.38 0.42 (%) Freeze/ Dimers 0.67 0.43 0.89 2.60 0.41 thaw (%)(−20° C.→ 5° C., 22 cycles)

It was found that the formation of dimers is inhibited by the additionof sucrose and trehalose.

Example 4 Influence of Sucrose on Heat Stability and Freeze/ThawStability

The influence of sucrose on heat stability and freeze/thaw stability wastested. Samples containing sucrose at various concentrations shown inTable 4 were prepared and tested as follows.

(1) Stability to thermal acceleration (50° C.-2W) was evaluated from theremaining hPM-1 percentage and the formation of multimers anddegradation products as determined by gel permeation chromatography(GPC). The number of insoluble particles per mL was measured by anautomatic light obscuration particle counter (HIAC).

(2) Stability to freeze/thaw cycling (3 cycles of storage at −20° C. for3 days and then 5° C. for one day) was evaluated from the remaininghPM-1 percentage and the formation of multimers and degradation productsas determined by gel permeation chromatography (GPC). The number ofinsoluble particles per mL was measured by an automatic lightobscuration particle counter (HIAC).

The results obtained are shown in Table 4. TABLE 4 <Test samples andresults> Sample Sample Sample Sample 16 17 18 19 hPM-1 (mg/mL) 20 20 2020 Sucrose (mg/mL) 0 25 50 100 Polysorbate 80 (mg/mL) 0.5 0.5 0.5 0.5Sodium Phosphate (mM) 15 15 15 15 pH 6.5 6.5 6.5 6.5 Initial hPM-1content (mg/mL) 19.2 19.2 19.3 19.3 Dimers (%) 0.18 0.16 0.15 0.15 Othermultimers (%) 0 0 0 0 Degradation products (%) 0 0 0 0 Number ofparticles of 0 0 12 0 10 μm or more (particles/mL) Number of particlesof 0 0 1 0 25 μm or more (particles/mL) Thermal Remaining hPM-1 (%) 98.298.5 97.8 97.8 acceleration Dimers (%) 1.37 1.47 1.36 1.41 (50° C.-2 W)Other multimers (%) 0 0 0 0 Degradation products (%) 0.92 0.89 0.89 0.89Number of particles of 0 0 0 0 10 μm or more (particles/mL) Number ofparticles of 0 0 0 0 25 μm or more (particles/ mL) Freeze/thaw RemaininghPM-1 (%) 100.2 100.8 100.4 100.2 (−20° C.→5° C., Dimers (%) 0.36 0.180.17 0.15 3 cycles) Other multimers (%) 0 0 0 0 Degradation products (%)0 0 0 0 Number of particles of 1 3 5 2 10 μm or more (particles/mL)Number of particles of 1 0 0 0 25 μm or more (particles/ mL)

It was found that the formation of dimers during freeze/thaw cycles isremarkably inhibited by the addition of sucrose. No variation instability with the concentration of sucrose was found.

Example 5 Influence of Antibody Concentration

The influence of the concentration of hPM-1 on heat stability wastested. Samples containing hPM-1 at various concentrations shown inTable 5 were prepared and tested as follows.

Stability to thermal acceleration (50° C.-2W) was evaluated from theremaining hPM-1 percentage and the formation of multimers anddegradation products as determined by gel permeation chromatography(GPC). The number of insoluble particles per mL was measured by anautomatic light obscuration particle counter (HIAC).

The results obtained are shown in Table 5. TABLE 5 <Test samples andresults> Sample Sample Sample 20 21 22 hPM-1(mg/mL) 17.5 20 22.5 Sucrose(mg/mL) 50 50 50 Polysorbate 80(mg/mL) 0.5 0.5 0.5 Sodium Phosphate(mM)15 15 15 pH 6.5 6.5 6.5 Initial hPM-1 content (mg/mL) 17.0 19.3 21.4Dimers (%) 0.16 0.16 0.18 Other multimers (%) 0 0 0 Degradation products0 0 0 (%) Number of particles 0 0 0 of 10 μm or more (particles/mL)Number of particles 0 0 0 of 25 μm or more (particles/mL) ThermalRemaining hPM-1 (%) 99.6 100.2 99.8 acceleration Dimers (%) 1.26 1.351.45 (50° C.-2 W) Other multimers (%) 0 0 0 Degradation products 0.950.93 0.99 (%) Number of particles of 0 3 0 10 μm or more (particles/mL)Number of particles of 0 0 0 25 μm or more (particles/mL)

No variation in stability with the concentration of hPM-1 was found.

Example 6 Influence of the Concentration of Phosphate Buffer

The influence of the concentration of phosphate buffer on heat stabilitywas tested. Samples containing phosphate buffer at variousconcentrations shown in Table 6 were prepared and tested as follows.

Stability to thermal acceleration (50° C.-2W) was evaluated from theremaining hPM-1 percentage and the formation of multimers anddegradation products as determined by gel permeation chromatography(GPC). The number of insoluble particles per mL was measured by anautomatic light obscuration particle counter (HIAC).

The results obtained are shown in Table 6. TABLE 6 <Test samples andresults> Sample Sample Sample 23 24 25 hPM-1(mg/mL) 20 20 20 Sucrose(mg/mL) 50 50 50 Polysorbate 80(mg/mL) 0.5 0.5 0.5 Sodium Phosphate(mM)10 15 20 pH 6.5 6.5 6.5 Initial hPM-1 content (mg/mL) 19.3 19.4 19.4Dimers (%) 0.17 0.18 0.18 Other multimers (%) 0 0 0 Degradation products0 0 0 (%) Number of particles 0 0 0 of 10 μm or more (particles/mL)Number of particles 0 0 0 of 25 μm or more (particles/mL) ThermalRemaining hPM-1 (%) 100.1 99.0 99.2 acceleration Dimers (%) 1.37 1.431.45 (50° C.-2 W) Other multimers (%) 0 0 0 Degradation products 0.940.95 0.94 (%) Number of particles 0 0 0 of 10 μm or more (particles/mL)Number of particles 0 0 0 of 25 μm or more (particles/mL)

No variation in stability with the concentration of phosphate buffer wasfound.

Example 7 Effects of Adding Sugars

Heat stability tests were performed to evaluate the effects of adding asugar (sucrose or mannitol) at anti-HM1.24 antibody concentrations inthe range of 2.5-10 mg/mL. Samples containing the sugar at variousconcentrations in low and high anti-HM1.24 antibody preparations (1 mL/5mL vial) and determined for the remaining percentage (%), multimers (%)and degradation products (%) under various storage conditions (60°C.-1W, 50° C.-3M, 5° C.-6M, Initial).

Test formulations of low-concentration preparations and the results areshown in Tables 7 and 8, while test formulations of high-concentrationpreparations and the results are shown in Tables 9 and 10. TABLE 7Sample Sample Sample Sample Sample 26 27 28 29 30 Sample 31 Sample 32Anti-H.M1.24 2.5 2.5 2.5 2.5 2.5 2.5 2.5 antibody (mg/mL) Sucrose 10 50100 — — — — (mg/mL) Mannitol — — — 10 50 100 — (mg/mL) NaCl (mM) 100 100100 100 100 100 100 pH 6.0 6.0 6.0 6.0 6.0 6.0 6.0

TABLE 8 Remaining Multimers Degradation percentage (%) (%) products (%)60° C.-1W Sample 26 90.9% 5.06% 1.99% Sample 27 91.1% 4.60% 1.98% Sample28 90.0% 4.14% 2.05% Sample 29 85.5% 5.04% 2.20% Sample 30 90.3% 4.99%1.99% Sample 31 86.6% 5.57% 2.63% Sample 32 88.9% 5.39% 2.09% 50° C.-3MSample 26 77.0% 14.0% 6.98% Sample 27 81.5% 13.7% 6.46% Sample 28 84.9%12.9% 4.83% Sample 29 78.9% 14.3% 7.31% Sample 30 75.2% 13.2% 6.72%Sample 31 76.1% 12.7% 6.24% Sample 32 76.8% 15.5% 7.62%  5° C.-6M Sample26 103.8% 3.82% 0.00% Sample 27 104.0% 3.44% 0.00% Sample 28 104.2%3.43% 0.00% Sample 29 103.8% 3.49% 0.00% Sample 30 104.3% 3.46% 0.00%Sample 31 104.3% 3.45% 0.00% Sample 32 103.5% 3.49% 0.00% Initial Sample26 100.0% 3.73% 0.00% Sample 27 100.0% 3.34% 0.00% Sample 28 100.0%3.34% 0.00% Sample 29 100.0% 3.38% 0.00% Sample 30 100.0% 3.36% 0.00%Sample 31 100.0% 3.36% 0.00% Sample 32 100.0% 3.38% 0.00%

After thermal acceleration at 50° C.-3M, the samples showed an increasein the remaining antibody monomer percentage and a decrease in theformation of multimers and degradation products in a manner dependent onthe concentration of sucrose added. After acceleration at 60° C.-1W, thesamples also showed a decrease in the amount of multimers formed. Underacceleration at 50° C.-3M, the effect of sugar addition on the remainingantibody percentage was more remarkable with sucrose than mannitol. Theeffect of sugar addition on the inhibition of association was also foundwith mannitol. TABLE 9 Sample Sample Sample Sample Sample Sample 33 3435 36 37 38 Anti-H.M1.24 2.5 5.0 5.0 10 10 10 antibody (mg/mL)Polysorbate 0.025 0.025 0.025 0.025 0.025 0.025 80 (%) Acetate (mM) 2020 20 20 20 20 NaCl (mM) 100 100 100 100 100 100 pH 6.0 6.0 6.0 6.0 6.06.0 Sucrose 10 10 20 10 40 0 (mg/mL)

TABLE 10 Remaining Multimers Degradation percentage (%) (%) products (%)60° C.-1W Sample 33 96.6% 4.78% 2.16% Sample 34 96.1% 6.47% 1.84% Sample35 96.1% 6.33% 1.84% Sample 36 96.1% 6.66% 1.76% Sample 37 97.0% 5.96%1.75% Sample 38 95.3% 7.11% 1.82% 50° C.-1M Sample 33 94.6% 5.01% 2.12%Sample 34 95.9% 5.62% 2.06% Sample 35 95.9% 5.27% 2.09% Sample 36 96.7%5.37% 1.97% Sample 37 97.1% 4.95% 1.96% Sample 38 95.5% 5.69% 2.02%  5°C.-6M Sample 33 107.8% 3.50% 0.00% Sample 34 106.1% 3.52% 0.00% Sample35 106.1% 3.51% 0.00% Sample 36 104.0% 3.59% 0.00% Sample 37 104.1%3.57% 0.00% Sample 38 103.7% 3.61% 0.00% Initial Sample 33 100.0% 3.40%0.00% Sample 34 100.0% 3.36% 0.00% Sample 35 100.0% 3.36% 0.00% Sample36 100.0% 3.38% 0.00% Sample 37 100.0% 3.37% 0.00% Sample 38 100.0%3.39% 0.00%

Comparison of the amounts of multimers formed after thermal accelerationshowed that association is inhibited better as the concentration ofsucrose added increases at the same concentration of anti-HM1.24antibody. It was found that sucrose also contributes to the inhibitionof association in high concentration anti-HM1.24 antibody formulations.

Example 8 Effects of Sugar Addition

Effects of sucrose addition were further tested at various amounts.Samples shown in Table 11 were prepared and stored at 50° C.-1M, afterwhich the remaining monomer antibody percentage and the amount ofmultimers were determined by GPC. The results obtained are shown inTable 12. TABLE 11 Sample 39 Sample 40 Sample 41 Sample 42 Anti-H.M1.2410 10 10 10 antibody (mg/mL) Polysorbate 80 (%) 0.05 0.05 0.05 0.05Acetate (mmol/L) 10 10 10 10 NaCl (mmol/L) 100 100 100 100 pH 6.0 6.06.0 6.0 Sucrose (mg/mL) 0 25 50 75

TABLE 12 Remaining percentage (%) Multimers (%) Initial 50° C.-1MInitial 50° C.-1M Sample 39 100.0% 83.3% 3.6% 12.2%  Sample 40 100.0%86.4% 3.6% 9.7% Sample 41 100.0% 87.8% 3.5% 8.4% Sample 42 100.0% 87.2%3.5% 8.9%

It was found that sucrose is effective for inhibiting the formation ofmultimers of anti-HM1.24 antibody.

Example 9 Effects of Adding Sugars (Freeze/Thaw Test)

The influence of adding sugars (nonreducing disaccharides andnonreducing trisaccharides) on freeze/thaw stability was tested. Samplescontaining sugars shown in Table 13 were prepared and subjected to afreeze/thaw test under the following conditions.

Stability to freeze/thaw cycling was evaluated from the formation ofdimers (multimers) as determined by gel permeation chromatography (GPC).<Test conditions> Thaw: −20° C. → 5° C. (1 hour) Hold:    5° C. (6hours) Freeze: 5° C. → −20° C. (1 hour) −20° C. (16 hours)

The above temperature cycle was repeated 3, 7 and 21 times. TABLE 13<Test samples and results> Sample Sample Sample Sample 43 44 45 46 hPM-1(mg/mL) 20 20 20 20 Polysorbate 80 (mg/mL) 0.5 0.5 0.5 0.5 SodiumPhosphate (mM) 15 15 15 15 pH 6.5 6.5 6.5 6.5 Additive (mM) — SucroseTrehalose Raffinose 145 145 145 Initial Dimers (%) 0.4 0.4 0.4 0.4 Othermultimers N.D. N.D. N.D. N.D. (%) Total amount of 0.4 0.4 0.4 0.4multimers (%)  3 Dimers (%) 0.7 0.4 0.5 0.8 freeze/ Other multimers N.D.N.D. N.D. N.D. thaw (%) cycles Total amount of 0.7 0.4 0.5 0.8 multimers(%)  7 Dimers (%) 0.8 0.5 0.4 1.0 freeze/ Other multimers N.D. N.D. N.D.N.D. thaw (%) cycles Total amount of 0.8 0.5 0.4 1.0 multimers (%) 21Dimers (%) 1.0 0.4 0.5 1.3 freeze/ Other multimers N.D. N.D. N.D. N.D.thaw (%) cycles Total amount of 1.0 0.4 0.5 1.3 multimers (%)

These results showed that the formation of dimers of hPM-1 antibodyduring freeze/thaw cycles can be remarkably inhibited by addingnonreducing disaccharides (sucrose, trehalose).

Example 10 Effects of Adding Sugars (Heat Stress Test)

The influence of adding sugars (nonreducing disaccharides andnonreducing trisaccharides) on stability during thermal loading wastested. Samples containing sugars shown in Tables 14 and 15 wereprepared and subjected to a heat stress test under the followingconditions.

Stability during thermal loading was evaluated from the formation ofdimers and multimers as determined by gel permeation chromatography(GPC). TABLE 14 <Test samples and results> Sample Sample Sample Sample47 48 49 50 hPM-1 (mg/mL) 20 20 20 20 Polysorbate 80 (mg/mL) 0.5 0.5 0.50.5 Sodium Phosphate (mM) 15 15 15 15 pH 6.5 6.5 6.5 6.5 Additive (mM) —Sucrose Trehalose Raffinose 145 145 145 Initial Dimers (%) 0.4 0.4 0.40.4 Other multimers N.D. N.D. N.D. N.D. (%) Total amount of 0.4 0.4 0.40.4 multimers (%) Heat Dimers (%) 5.2 6.0 5.6 6.9 stress Other multimers6.1 4.5 4.5 4.7 test (%) 60° C.- Total amount of 11.2 10.5 10.0 11.7 14days multimers (%)

These results showed that the total amount of multimers and theformation of other multimers in hPM-1 antibody formulations can beremarkably inhibited by adding nonreducing disaccharides (sucrose,trehalose). TABLE 15 Sample Sample Sample Sample 51 52 53 54 Anti-HM1.24antibody 10 10 10 10 (mg/mL) Polysorbate 80 (mg/mL) 0.25 0.25 0.25 0.25Acetate (mM) 30 30 30 30 pH 6.0 6.0 6.0 6.0 Additive (mM) — SucroseTrehalose Raffinose 145 145 145 Initial Dimers (%) 2.8 2.8 2.8 2.8 Other0.5 0.5 0.5 0.5 multimers (%) Total amount 3.3 3.3 3.3 3.3 of multimers(%) Heat Dimers (%) 9.2 10.4 9.5 9.9 stress Other 5.6 2.9 4.1 4.3 testmultimers (%) 60° C.- Total amount 14.8 13.3 13.6 14.2 14 days ofmultimers (%)

It was shown that the total amount of multimers and the formation ofother multimers are remarkably inhibited by adding nonreducingdisaccharides (sucrose, trehalose) in anti-HM1.24 antibody formulationssimilarly to hPM-1 antibody formulations.

Example 11 Effects of Adding Sugars (Light Acceleration Test)

The influence of adding sugars (nonreducing disaccharides andnonreducing trisaccharides) on stability during light acceleration wastested. Samples containing sugars shown in Tables 16 and 17 wereprepared and subjected to a light acceleration test under the followingconditions.

Stability during light acceleration was evaluated from the formation ofdimers and multimers as determined by gel permeation chromatography(GPC). TABLE 16 <Test samples and results> Sample Sample Sample Sample55 56 57 58 hPM-1 (mg/mL) 20 20 20 20 Polysorbate 80 (mg/mL) 0.5 0.5 0.50.5 Sodium Phosphate (mM) 15 15 15 15 pH 6.5 6.5 6.5 6.5 Additive (mM) —Sucrose Trehalose Raffinose 145 145 145 Initial Dimers (%) 0.4 0.4 0.40.4 Other N.D. N.D. N.D. N.D. multimers (%) Total amount 0.4 0.4 0.4 0.4of multimers (%) Light Dimers (%) 3.5 2.5 3.2 3.5 acceleration OtherN.D. N.D. N.D. N.D. test multimers (%) 1,200,000 Total amount 3.5 2.53.2 3.5 Lux · hr of multimers (%)

It was shown that the light-induced dimerization of hPM-1 antibody canbe remarkably inhibited by adding sucrose. TABLE 17 Sample Sample SampleSample 59 60 61 62 Anti-HM1.24 antibody 10 10 10 10 (mg/mL) Polysorbate80 (mg/mL) 0.25 0.25 0.25 0.25 Acetate (mM) 30 30 30 30 pH 6.0 6.0 6.06.0 Additive (mM) — Sucrose Trehalose Raffinose 145 145 145 InitialDimers (%) 2.8 2.8 2.8 2.8 Other 0.5 0.5 0.5 0.5 multimers (%) Totalamount 3.3 3.3 3.3 3.3 of multimers (%) Light Dimers (%) 3.8 4.1 3.4 3.1acceleration Other 2.8 0.8 2.8 2.9 test multimers (%) 1,200,000 Totalamount 6.6 4.9 6.2 6.0 Lux.hr of multimers (%)

It was shown that the light-induced association of anti-HM1.24 antibodycan be remarkably inhibited by adding sucrose.

Example 12 Effects of Adding Surfactant Species

The influence of surfactant species on freeze/thaw stability was tested.Samples containing surfactants shown in Table 18 were prepared andtested as follows.

Stability to freeze/thaw cycling (3 cycles of freeze at −25° C./thaw at4° C.) was evaluated from the number of particles per mL as measured byan automatic light obscuration particle counter (HIAC). TABLE 18 <Testsamples and results> Sample Sample Sample Sample Sample Sample SampleSample Sample Sample Sample Sample 63 64 65 66 67 68 69 70 71 72 73 74HPM-1 (mg/mL) 4 4 4 4 4 4 4 4 4 4 4 4 NaCl (mM) 250 250 250 250 250 250250 250 250 250 250 250 Sodium Phosphate (mM) 20 20 20 20 20 20 20 20 2020 20 20 pH 7.0 7.0 7.0 7.0 7.0 7.0 7.0 7.0 7.0 7.0 7.0 7.0 Polysorbate80 (mg/mL) 0 0.005 0.01 0.05 0.1 0 0 0 0 0 0 0 Polysorbate 20 (mg/mL) 00 0 0 0 0.01 0.05 0.1 0 0 0 0 Poloxamer 188 (mg/mL) 0 0 0 0 0 0 0 0 0.10.5 1 2 Freeze/thaw Number of 290 49 22 9 9 14 15 8 7 6 4 5 (−25° C.→4°C., particles of 3 cycles) 10 μm or more (particles/mL) Number of 13 0 11 0 2 3 3 2 2 0 2 particles of 25 μm or more (particles/mL)

It was found that the formation of insoluble particles duringfreeze/thaw cycles is remarkably inhibited by the addition of surfactantspecies (Polysorbate 80, Polysorbate 20, Poloxamer 188).

1. An antibody-containing solution formulation including a sugar as astabilizer.
 2. The solution formulation of claim 1 further including asurfactant as a stabilizer.
 3. The solution formulation of claim 1wherein the sugar is a sugar alcohol or a nonreducing oligosaccharide.4. The solution formulation of claim 1 wherein the sugar is anonreducing oligosaccharide.
 5. The solution formulation of claim 1wherein the sugar is mannitol, sucrose, trehalose or raffinose.
 6. Thesolution formulation of claim 1 wherein the sugar is sucrose, trehaloseor raffinose.
 7. The solution formulation of claim 1 wherein the sugaris sucrose or trehalose.
 8. The solution formulation of claim 1 whereinthe sugar is sucrose.
 9. The solution formulation of claim 2 wherein thesurfactant is Polysorbate 80 or
 20. 10. The solution formulation ofclaim 1 wherein the antibody is a recombinant antibody.
 11. The solutionformulation of claim 10 wherein the antibody is a chimeric antibody,humanized antibody or human antibody.
 12. The solution formulation ofclaim 1 wherein the antibody is an IgG class antibody.
 13. The solutionformulation of claim 12 wherein the IgG class antibody is an IgG1 classantibody.
 14. The solution formulation of claim 1 wherein the antibodyis an anti-interleukin-6 receptor antibody or anti-HM1.24 antibody. 15.A method for inhibiting the formation of antibody multimer molecules inan antibody-containing solution formulation, comprising adding a sugarinto the solution.
 16. A method for inhibiting the formation of antibodymultimer molecules during freeze/thaw cycles of an antibody-containingsolution, comprising adding a nonreducing oligosaccharide into thesolution.
 17. A method for inhibiting the formation of antibody multimermolecules during freeze/thaw cycles of an antibody-containing solution,comprising adding a nonreducing disaccharide or nonreducingtrisaccharide into the solution.
 18. A method for inhibiting theformation of insoluble particles during freeze/thaw cycles of anantibody-containing solution, comprising adding a surfactant.
 19. Amethod for stabilizing an antibody during freeze/thaw cycles of asolution containing the antibody, comprising adding a nonreducing sugarand a surfactant.